The ultimate goal of this application is to develop a novel ex vivo purging method using myxoma virus (MYXV), a rabbit-specific poxvirus, to improve the clinical outcomes in treatment of multiple myeloma (MM). MM is a clonal plasma cell malignancy that has to date resisted essentially all therapeutic strategies. Currently, the standard of care for patients with MM is treatment with high-dose chemotherapy followed by autologous stem cell transplantation (ASTC). Although ASCT can often increase the disease-free interval for eligible patients, the disease generally relapses. This cancer relapse is mediated by cells derived from one or both of two sources: myeloma cells remaining within patient's hematopoietic system that resist the chemotherapy, and/or residual myeloma cells that contaminate the autologous stem cell graft sample. Recently, MYXV, when used to pre-treat primary MM patient ASTC samples, was found to be able to delete all residual MM cells while completely sparing the normal human stem cells needed to reconstitute the recipient immune system in a xenotransplantation animal model. MYXV is completely nonpathogenic to humans or mice, but has a natural tropism for a variety of human cancer cells and is being developed as a viral oncolytic agent for the treatment of a variety of cancers. We propose that ex vivo MYXV treatment of ASCT grafts prior to transplant will reduce MM disease relapse rates. In this Phase I STTR application, three specific aims are proposed to progress the development of MYXV as a safe therapeutic drug for the ex vivo purging of cancer cells to be used before ASCT in MM patients: (1) Evaluate safety by in vitro testing the interaction of MYXV with primary human transplant bone marrow cells and peripheral blood mononuclear cells. (2) To assess the specific MYXV vector clone chosen for clinical development and the purging methodology in an appropriate animal model. The intention of this Aim is to replicate in toto the exact clinical ex vivo purging strategy that will be proposed to the FDA for the human Phase I clinical trial. (3) Optimize the procedures for the ex vivo treatment of human bone marrow or peripheral blood mononuclear cells with MYXV to provide preclinical data required to support a future human clinical safety trial. The various factors to be optimized include the formulation for the MYXV stocks that ensures product stability and is compatibility with ex vivo treatment, the incubation buffer and conditions, the ratio of MXYV particles or infectious units to total nucleated cells in the transplant, the concentration of virus and cells during ex vivo incubation, and the minimum and maximally effective and no- effect doses of MYXV.